Genetic studies indicate that mutations in the PrP gene may contribute to the course of the disease in infected animals. Perhaps most excitingly it appears that genetic changes in the PrP ORF may also perhaps promote its de novo appearance. This study intends to examine the role of PrP in scrapie by mutational analysis. The first objective is the construction of libraries of randomly mutated hamster and mouse PrP genes. Targeted random mutagenesis will be used to produce libraries of mutant hamster and mouse PrP genes. The ORFs will be divided into arbitrary domains and a separate library will be constructed for each section of the polypeptide coding region. The ORF cassettes used will be epitopically "tagged" to allow discrimination of the exogenous recombinant protein from endogenous cellular PrP. The pSPOXII.neo vector will be used to express these mutant genes in both infected and uninfected murine and hamster cells in culture. Two distinct strategies will be employed. Firstly a representative catalog of mutants will be assembled. These mutants will be characterized by sequencing to define the mutation and will be tested in vivo for scrapie-specific activity following lipofectin mediated transfection into scrapie-infected or uninfected mouse and hamster cells in culture. In addition pools of randomly mutated hamster and mouse ORFs will be expressed in both infected and uninfected mouse N2a and Syrian hamster SH cells. Mutations which affect the eligibility of PrP for conversation to the protease-resistant for, as well as mutants that affect the heterologous gene directed PrPSc "blocking" activity will be identified and defined by DNA sequencing. These assays will be performed on a representative cross-section of mutant PrP genes. In addition, providing that a suitable in situ colony assay can be developed, pools of randomly selected from the library will be screened by in situ immunoassay of colonies replicated onto polyester or nitrocellulose filters. In addition, we shall screen for mutations which can initiate the de novo appearance of PrPSc and/or infectious prions in cultured cells will be screened for. These assays will also be performed on both individual, characterized clones as well as on pools of random clones. In addition, bioassays will be performed on partially purified extracts of transfectant cells.